Journal: IBRO Neuroscience Reports
Article Title: An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation
doi: 10.1016/j.ibneur.2022.09.004
Figure Lengend Snippet: An alternative splice isoform of CDK5RAP2. (A) mRNA structure of mouse CDK5RAP2. CM1 domain refers to the centrosomin N-terminal motif 1, which binds to and activates γTuRC. The C-terminal domain is responsible for the localization of CDK5RAP2 at the centrosome and at the Golgi apparatus. Primers used for RT-PCR were shown. (B) RT-PCR analysis on the exon 17 skipping. Total RNA from embryonic day 13.5 brain, postnatal day 1 (P1D), and postnatal day 7 cerebrum were analyzed with primers flanking exon 17 (Pr1 and Pr5). Skipping of exon 17 (Δe17) was detected after P1D. (C) Amplicons with primer sets used for real time-RT-PCR shown in (D). DNAs were eluted from the upper and the lower bands of the gel shown in (B) and were amplified with primers specific to each isoform. (D) Quantification of the expression of CDK5RAP2 isoforms measured with real-time RT-PCR. The full-length isoform was amplified with primer set Pr2 and Pr4, and the Δe17 isoform was amplified with primer set Pr3 and Pr4. Error bars are S. E. (E) Real-time PCR analysis on the change in the isoform expression during the culture of primary cortical neurons. DIV stands for days in vitro. (F) Detection of CDK5RAP2 proteins in tissues. Lysates from the P7D cerebrum and P21D testis were immunoprecipitated with anti-CDK5RAP2 antibody and immunoblotted with the same antibody. The star indicates the band with a molecular weight close to the calculated molecular weight of Δe17 isoform.
Article Snippet: Antibodies and their dilutions were as follows: rabbit polyclonal antibody against pericentrin (Covance #923701, Burlington, NC) [1:500], rabbit polyclonal antibody against CDK5RAP2 (MILLIPORE #06–1398, Burlington, MA) [1:500], mouse monoclonal antibody against α-tubulin (Sigma-Aldrich #T5168, St. Louis, MO) [1:500], rabbit polyclonal antibody against α-tubulin (Thermo Fisher Scientific #RB-9281, Waltham, MA) [1:200], mouse monoclonal antibody against acetylated tubulin (Sigma-Aldrich #T6793) [1:500], goat polyclonal antibody against γ-tubulin (Santa Cruz #sc-7396, Santa Cruz, CA) [1:500], mouse monoclonal antibody against FLAG (clone M2; Sigma-Aldrich) [1:1000] and mouse monoclonal antibody against GM130 (BD Biosciences #610822, Franklin Lakes, NJ) [1:400].
Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Immunoprecipitation, Molecular Weight
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